1. Field of the Invention
The present invention relates to a method for qualitatively or quantitatively analyzing a target RNA having a specific nucleotide sequence, which is considered to be contained in a gene mixture of DNA, RNA, or the like, and the method is useful in the field of clinical diagnoses, such as gene diagnosis and the like. Also, the present invention relates to a method for qualitatively or quantitatively analyzing microorganisms in environments, such as foods, indoors, soils, rivers, ocean, and the like.
2. Discussion of the Background
Generally, a high specificity and a high sensitivity are required for an assay of biological components. An assay of a nucleic acid having a specific nucleotide sequence (target nucleic acid) can use a property that the nucleic acid sequence-specifically forms a complex with another nucleic acid having a sequence complementary to the specific nucleotide sequence (nucleic acid probe).
When the target nucleic acid having a specific nucleotide sequence is assayed, a means for obtaining a measurable signal related to the amount of the formed complex is important. Furthermore, the amount of the target nucleic acid which can be present in a sample is extremely small for a purpose of clinical diagnosis etc. so that such a means requires a step for amplifying the extremely small amount of the nucleic acid.
In diagnosis of viral infection, since the amount of a target nucleic acid (viral nucleic acid) in a clinical sample is often extremely small, a polymerase chain reaction (PCR), particularly a competitive PCR, is known as a means for obtaining a high sensitivity by improving the signal strength in order to realize the measurement with high sensitivity and good reproducibility. In this method, PCR is carried out by adding a competitor (a different nucleic acid sequence having a primer recognizing region common to the target nucleic acid) having a known concentration to a sample, and the concentration of the target nucleic acid in the sample is estimated by comparing amplified degrees between the competitor and the target nucleic acid. More specifically, a nucleic acid having a sequence complementary to a primer on its terminus and can be distinguished from the amplified product of the target nucleic acid by a separation means, such as electrophoresis or the like (e.g., based on a different chain length) is prepared, this nucleic acid is added to a sample to give respective concentrations, and PCR of these mixtures is simultaneously carried out.
Additionally, an assay method in a homogenous system has been proposed as a method for assaying a target nucleic acid using PCR. For example, an assay method has been proposed, in which PCR is carried out in the presence of an intercalating fluorochrome, the fluorescence of the reaction solution is measured in each PCR cycle, and the initial amount of the target nucleic acid is determined based on its changes (JP-A-5-237000 (the term xe2x80x9cJP-Axe2x80x9d as used herein means an xe2x80x9cunexamined published Japanese patent applicationxe2x80x9d); Igaku-no Ayumi, 173(12): 959-963 (1995); Analytical Biochemistry, 229: 207-213 (1995)). In this assay method, the amplified product by PCR is a double-stranded DNA so that an intercalating fluorochrome having a property to change its fluorescence characteristic, such as an increase in the fluorescence intensity by intercalation into the double-stranded nucleic acid, is used, the fluorochrome is added to the reaction solution in advance before amplification operation by PCR, the fluorescence intensity in the reaction solution is measured with time, and the initial amount of the target nucleic acid is determined, for example, based on its build up cycle etc.
On the other hand, NASBA method and 3SR method are also known as RNA amplification methods. In these methods, a double-stranded DNA fragment containing a promoter sequence is synthesized for a target RNA using a primer containing the promoter sequence, a reverse transcriptase and ribonuclease H, an RNA containing a specific nucleotide sequence of the target RNA is synthesized in the presence of an RNA polymerase, and then a chain reaction is carried out in which the RNA is subsequently used as the template for the synthesis of the double-stranded DNA containing the promoter sequence. Thereafter, the reaction is completed when the RNA is amplified, and the amplified RNA is determined by an electrophoresis method or a hybridization method using a labeled nucleic acid probe.
In the hybridization method using a labeled nucleic acid probe, a nucleic acid probe labeled in such a manner that it generates a measurable signal, such as visible light, fluorescence, emission, or the like, forms a complex with a target nucleic acid, and then unreacted nucleic acid probe is washed or degraded, and the label is measured to assay the target nucleic acid. A method called sandwich assay is generally known, which uses two probes each comprising a sequence capable of forming a complementary bond with a specified sequence at different region in a specified nucleic acid. In this method, a first probe is immobilized on an insoluble carrier, and a part of a second probe is labeled with a dye having a color in the visible region, a fluorescent material, or an enzyme capable of forming them. Then, these probes are added to a sample, a specified nucleic acid in the sample is complementary bound to the first and second probes, and a complex composed of these three materials is formed on the insoluble carrier. Subsequently, the resultant supernatant in the sample reaction solution is separated from the insoluble carrier to separate the free second probe (B/F separation step). Thereafter, the presence or absence and amount of the specified nucleic acid in the sample are determined by measuring the label in the complex on the insoluble carrier. Also, when an enzyme which can form a dye having a color in the visible region or a fluorescent material is used as the second probe, the free second probe is removed after the complex forming step, an enzyme substrate as the precursor is added to the sample reaction solution, and then the presence or absence and amount of the target nucleic acid in the sample are determined by, measuring the dye or fluorescent material which is the reaction product.
Since the sandwich assay uses an insoluble carrier in the reaction solution, the second probe is nonspecifically adsorbed on the insoluble carrier. Accordingly, when the label in the complex on the insoluble carrier is measured, an error occurs in the measured results due to the presence of the label of the second probe nonspecifically adsorbed on the insoluble carrier. Thus, a problem is occurred when the presence or absence and amount of the specified nucleic acid in the sample are determined. Particularly, since the diagnosis of viral infection requires detection of an extremely small amount of viral nucleic acid in a clinical sample with a good reproducibility and a high sensitivity, the problem caused by nonspecific adsorption is an important problem to be solved.
In order to avoid this problem, various attempts are made, such as hydrophilic treatment of the surface of the insoluble carrier, blocking of adsorption points of the carrier surface with a protein etc., sufficiently washing of the insoluble carrier after the B/F separation step, and the like.
However, in the chemical hydrophilic treatment of the carrier surface, its result depends on the material of the carrier, and it is not always easy technically. Also, in the method in which the carrier surface is coated with a protein in order to block adsorption points on the carrier surface in advance, there is a possibility that the protein interacts with the nucleic acid moiety or label of the second probe to cause additional nonspecific adsorption on the carrier. Additionally, in the B/F separation step, there is an operational limitation in increasing the number of times of washing. Thus, for example, when a surfactant is added to the washing solution, degradation of the complex formed on the carrier may be accelerated.
In the competitive PCR method, in order to assay one test sample, it is necessary to prepare a competitor having various concentrations including an assumed nucleic acid concentration and carry out PCR of a sample to which the competitor has been added. Additionally, an separation operation, such as electrophoresis or the like, must be carried out by taking out the sample from the reaction container after completion of PCP. Accordingly, it is inadequate to applying it to clinical tests in which a large number of samples must be treated quickly and conveniently. Also, since the sample must be taken out from the reaction solution, a problem of causing a pseudopositive reaction due to scattering of the amplified product cannot be solved. Additionally, when the target is RNA, so-called RT-PCR in which PCR is carried out after once synthesizing a cDNA using the RNA as the template in the presence of a reverse transcriptase must be carried out so that it is necessary substantially to carry out two stage steps.
In the method in which PCR is carried out in the presence of an intercalating fluorochrome, its principle is intercalation into a double-stranded nucleic acid. Accordingly, when double-stranded DNAs contaminant other than the specified nucleic acid, such as a large amount of genomic DNA and the like, are present in the test sample, the intercalating fluorochrome is also intercalated into them to cause a problem of generating a large background. Also, in the PCR method, a pair of complementary oligonucleotides of the specified nucleic acid sequence are used as elongation reaction primers, but they mutually form a complementary bond depending on the primer sequences and, as a result, sometimes produce a primer dimer mutually using the other primer as the template. Since the intercalating fluorochrome nonspecifically intercalates into double-strands, it also causes a problem in that the background increases due to the production of such a dimer.
When the NASBA method or 3SR method is used, it is possible to determine the amplified nucleic acid by an electrophoresis method or a hybridization method using a labeled nucleic acid probe after the reaction after amplification of the RNA to a sufficiently detectable amount. However, it is impossible to determine the target RNA originally present in the sample before the amplification. Also, when the electrophoresis method or the hybridization method using a labeled nucleic acid probe is carried out, there is a problem in that a pseudopositive reaction occurs due to scattering of the amplification product.
Accordingly, an object of the present invention is to provide a quick, convenient and highly accurate method for assaying a target RNA (single-stranded RNA) containing a specified nucleic acid sequence originally present in the sample, and particularly, to provide a method by which the entire operations can be completed in a closed container.
Specifically, the present invention relates to the following (1) to (7).
(1) A method for assaying a target nucleic acid, comprising:
providing an RNA amplification system comprising:
producing a double-stranded DNA using, as a template, a target RNA containing a specific nucleotide sequence in a sample, said double-stranded DNA having a promoter sequence and being capable of transcribing an RNA comprising the specific nucleotide""sequence or a sequence complementary to the specific nucleotide sequence;
producing an RNA transcription product comprising the specific nucleotide sequence or a sequence complementary to the specific nucleotide sequence in the presence of an RNA polymerase; and
producing the double-stranded DNA using the RNA transcription product as a template, in the presence of a probe labeled with an intercalating fluorochrome having a sequence, complementary to the RNA transcription product;
measuring the fluorescence intensity in the RNA amplification system with time;
calculating a time when the fluorescence intensity satisfies a prescribed criterion based on the measured change in the fluorescence intensity with time; and
determining a concentration of the target nucleic acid in the sample based on the calculated time
(2) The method according to the above (1), wherein the time which satisfies a prescribed criterion is a time to reach a definite fluorescence intensity.
(3) The method according to the above (1), wherein the time which satisfies a prescribed criterion is a time when an increasing rate of the fluorescence intensity per unit of time becomes a maximum.
(4) The method according to any one of the above (1) to (3), wherein the concentration of the target nucleic acid is determined by comparing the time which satisfies a prescribed criterion with the times in at least two RNA samples containing the specific nucleotide sequence at a different concentration.
(5) The method according to the above (1), wherein, in the RNA amplification system,
a single-stranded DNA is produced in the presence of an RNA-dependent DNA polymerase using a primer having a sequence complementary to the specific nucleotide sequence and a primer having a homologous sequence of the specific nucleotide sequence, wherein one of the primers is a promoter primer having a promoter sequence of the RNA polymerase at the 5xe2x80x2-side, and using the target RNA as a template;
the double-stranded DNA is produced in the presence of a DNA-dependent DNA polymerase using the single-stranded DNA as a template;
the RNA transcription product is produced from the double-stranded DNA in the presence of a RNA polymerase; and
the RNA transcription product is subsequently used as the template for producing the single-stranded DNA in the presence of the RNA-dependent DNA polymerase.
(6) The method according to the above (1), wherein a complex formed by a complementary bond between the probe labeled with an intercalating fluorochrome and the RNA transcription product has a fluorescence characteristic different from the probe labeled with an intercalating fluorochrome which is not form a complex with the RNA transcription product.
(7) The method according to the above (1) or (6), wherein the probe labeled with an intercalating fluorochrome complementary bonds to the RNA transcription product so that the intercalating fluorochrome is intercalated between the produced RNA and the probe.